More than half of all human cancers are associated with one or more alterations in the tumor suppressor gene TP53 (1-4). Many premalignant lesions, a subset of malignant clones, and germlines of families prone to cancer are characterized by the presence of one wild-type and one mutant allele of TP53 (5-9). In this situation the mutant p53 protein may act in a dominant-negative fashion, ultimately leading to loss of heterozygosity and thus a further growth advantage for the malignant cells. Alternatively, the mutant p53 protein may have acquired a new tumor promoting activity which is independent of wild-type p53. These hypotheses are based on the analysis of only a few TP53 mutations usually in the setting of over-expression of the mutant protein, and their relevance to TP53 mutations in general has not been proven (8, 10-13).
There is a need in the art for additional systems in which to study mutations in human p53, an important and ubiquitous cancer suppressor gene.
It is an object of the invention to provide a yeast cell useful for selecting and studying mutations in human p53.
It is another object of the invention to provide a method of identifying compensatory mutations in TP53 which suppress dominant-negative TP53 mutant phenotypes.
It is still another object of the invention to provide a method of identifying potential therapeutic agents.
It is yet another object of the invention to provide a method of screening putative carcinogens.
It is yet another object of the invention to provide a method for identifying cellular proteins which interact with p53 and abrogate its activity.
It is an object of the invention to provide a kit for isolating mutations in p53.
It is another object of the invention to provide a gene fusion useful for isolating and studying p53 mutations.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention a yeast cell is provided. The cell comprises a first reporter gene which is selectable or counterselectable. The reporter gene is operably linked to a DNA sequence to which human p53 specifically binds. The cell also comprises a first fusion gene which expresses a human p53 in the cell. The fusion gene comprises a promoter operably linked to a human p53 coding sequence.
In another embodiment of the invention a method of identifying compensatory mutations in TP53 which suppress dominant-negative TP53 mutant phenotypes is provided. The method involves providing a cell which comprises:
(i) a reporter gene which is selectable, wherein the reporter gene is operably linked to a DNA sequence to which human p53 specifically binds; and
(ii) a first fusion gene which expresses a dominant-negative allele of human p53 in the cell, the fusion gene comprising a promoter operably linked to a human p53 coding sequence.
Then a population of DNA molecules comprising a second fusion gene is introduced into the cell. The second fusion gene comprises a promoter operably linked to a mutagenized human p53 coding sequence. Phenotypic revertants of the dominant-negative allele of human TP53 are selected using the selectable phenotype of the reporter gene.
According to another embodiment of the invention a method of identifying potential therapeutic agents is provided. A cell is provided which comprises:
a reporter gene which is selectable, wherein the reporter gene is operably linked to a DNA sequence to which human p53 specifically binds; and
a fusion gene which expresses a dominant-negative allele of human TP53 in the cell, the fusion gene comprising a promoter operably linked to a human p53 coding sequence.
Test compounds are contacted with the cell. The selectable phenotype of the reporter gene is assayed. Desirable test compounds are identified as potential therapeutic agents if they induce the cell to display the selectable phenotype.
In another aspect of the invention a method of screening putative carcinogens for their effect on a p53 allele is provided. A cell is provided which comprises:
a reporter gene which is counterselectable, wherein the reporter gene is operably lied to a DNA sequence to which human p53 specifically binds; and
a fusion gene which expresses human p53 in the cell, the fusion gene comprising a promoter operably linked to a human p53 coding sequence.
The cell is contacted with a putative carcinogen. Cells are isolated which contain a mutation in the human p53 coding sequence by counterselecting for loss of expression of the reporter gene.
According to another embodiment of the invention cellular proteins which interact with p53 and abrogate its activity are identified. A population of cells is provided which comprise:
a reporter gene which is counterselectable, wherein the reporter gene is operably linked to a DNA sequence to which human p53 specifically binds; and
a fusion gene which expresses human p53 in the cell, the fusion gene comprising a promoter operably linked to a human p53 coding sequence.
A library of human cDNA molecules is introduced into the population of cells. Each of the cDNA molecules is operably linked to expression control sequences so that the human cDNA is expressed in the cell. The cells are assayed to identify those which express the counterselectable phenotype of the reporter gene. The counterselectable phenotype identifies cells which express a protein which abrogates p53 activity.
According to another aspect of the invention a kit is provided. The kit comprises three yeast strains. The first yeast strain comprises a centromeric plasmid which itself comprises: a fusion of a yeast alcohol dehydrogenase promoter operably linked to a human p53 coding sequence; and a yeast histidine (HIS3) gene. The first yeast strain also comprises an integrated reporter gene which consists of a p53 consensus binding sequence inserted upstream from the URA3 locus. The second yeast strain comprises an integrated reporter gene which consists of a p53 consensus binding sequence inserted upstream from the URA3 locus. The third yeast strain comprises a centromeric plasmid which itself comprises a fusion of a yeast alcohol dehydrogenase promoter operably linked to a human p53 coding sequence, and a yeast LEU2 gene. The third yeast strain also contains an integrated reporter gene which consists of a p53 consensus binding sequence inserted upstream from the URA3 locus. The first strain is of a compatible mating type to the second and third strains.
In still another embodiment of the invention a tripartite gene fusion is provided. The fusion comprises a human p53-specific DNA-binding site; a yeast URA3 gene; and a portion of a yeast SPO13 gene. The human p53-specific DNA-binding site is upstream of the URA3 gene, and the portion of the yeast SPO13 gene is interposed between the URA3 gene and the human p53-specific DNA-binding site. Moreover, the portion of the yeast SPO13 gene consists of the first 15 codons of SPO13 and nucleotides 5xe2x80x2 to nucleotide xe2x88x92170.
These and other embodiments of the invention provide the art with tools for studying mutagenesis and carcinogenesis in general, as well as for studying the important cancer-related gene TP53.